Use of peptides for promoting wound healing

ABSTRACT

The current invention relates to the use of a peptide comprising an amino acid sequence in the preparation of a medicament for the regeneration of tissue, preferably for the treatment of a wound. Further the invention relates to compositions comprising such peptides, and use of said peptides in both medical and nonmedical (cosmetic) applications.

The current invention relates to the use of a peptide in the preparationof a medicament for the regeneration of tissue, for the treatment ofskin, and/or for the treatment of a wound. Further the invention relatesto such peptides and compositions comprising them, and to the use ofsaid peptides in both medical and non-medical (cosmetic) applications.

Wounds inevitably happen during the lifetime of any animal being, andmay be the consequence of a wide variety of occurrences, like contactwith sharp or hot objects, or are seen in certain clinical conditionslike diabetes.

Another clinical important example is the development of decubitus(bedsores), i.e. lesions caused by unrelieved pressure to any part ofthe body, especially portions over bony or cartilaginous areas. Althoughcompletely treatable if found early, without medical attention,bedsores, like any wound, can become life threatening.

In case of general reduced health status, the closure (healing) ofwounds may be delayed, and can give rise to additional problems likeinfections, inflammation, tissue necrosis, and non-efficient woundclosure becomes, again, life threatening.

It is not surprising much research has been directed to understandingthe mechanisms that are important in the closure (healing) of wounds andthe repair of damaged tissue, including damaged skin, as it isinevitable that rapid closure of wound is of importance for the humanand animal health.

Indeed there is nowadays increased understanding of the mechanismsinvolved (see for example the excellent review by Martin et al, Science,Vol 276, 75 (1997)).

In general, wound healing is described as consisting of 3 phases, i.e.the inflammatory phase, the proliferative phase, and the maturationalphase (referred to as acute inflammatory phase, extracellular matrix andcollagen synthesis, and remodeling (Peacock, E. E., Jr., Wound Repair,2nd edition, W B Saunders, Philadelphia (1984)).

The sequence of the healing process is initiated during an acuteinflammatory phase with the deposition of provisional tissue. This isfollowed by re-epithelialization, collagen synthesis and deposition,fibroblast proliferation, and neovascularisation, all of whichultimately define the remodeling phase (Clark, R. A. F., J. Am. Acad.Dermatol. 13:701 (1985)).

These events are influenced by growth factors and cytokines secreted byinflammatory cells or by the cells localized at the edges of the wound(Assoian, R. K. et al., Nature (Lond.) 309:804 (1984); Nemeth, G. G. etal., “Growth Factors and Their Role in Wound and Fracture Healing,”Growth Factors and Other Aspects of Wound Healing in Biological andClinical Implications, New York (1988), pp. 1-17.

The inflammatory phase is characterized by haemostasis and inflammation.Collagen exposed during wound formation activates the clotting cascade(both the intrinsic and extrinsic pathways), initiating the inflammatoryphase. Platelets, the first response cell, release multiple chemokinesthat help stabilize the wound through clot formation. These mediatorsact to control bleeding and limit the extent of injury. The secondresponse cell to migrate to the wound, the neutrophil, is responsiblefor debris scavenging, complement-mediated opsonisation of bacteria, andbacteria destruction via oxidative burst mechanisms (i.e. superoxide andhydrogen peroxide formation). The macrophage is essential for woundhealing. Numerous enzymes and cytokines are secreted by the macrophage,which marks the transition into the process of tissue reconstruction,i.e., the proliferative phase.

During the proliferative phase, epithelialization, angiogenesis,granulation tissue formation, and collagen deposition are the principalsteps in wound healing. Epithelialization occurs early in wound repair.Angiogenesis, stimulated by for example TNF-alpha, is marked byendothelial cell migration and capillary formation. The new capillariesdeliver nutrients to the wound and help maintain the granulation tissuebed. The final part of the proliferative phase is granulation tissueformation. Fibroblasts differentiate and produce ground substance andthen collagen. The ground substance is deposited into the wound bed;collagen is then deposited as the wound undergoes the final phase ofrepair. Many different cytokines, including PDGF, insulin like growthfactor (IGF), and EGF are involved in the proliferative phase of woundrepair. During the maturational phase the wound undergoes contraction,ultimately resulting in a smaller amount of apparent scar tissue.

It will be obvious from the above that proper wound healing involves acomplex interaction of cells and substances like cytokines working inconcert. Based on fundamental research, many drugs, substances andmethods of treatment have been proposed to stimulate wound healing.

Indeed, manipulation of the healing process through woundsupplementation with agents that are (natural) contributors to thehealing process is an appealing concept. Early experimental studiesevaluating wounds supplemented with inflammatory mediators usedmaterials extracted from cell preparations and generated encouragingresults (see for example Clin Plast Surg. 2007 October; 34(4):659-71.)Recombinant technology has allowed the production of larger volumes ofthese mediators that can be used more practically and safely in theclinical setting.

Typical examples of such substances include for example Regranex(Becaplermin; a genetically engineered recombinant PDGF; Johnson &Johnson) Inc), a medicine that contains a platelet-derived growth factor(PDGF) and is indicated for the treatment of deep neuropathic diabeticfoot ulcers.

Another example is the use of FGF's, for example FGF-2 (e.g. Curr DrugDeliv. 2006 October; 3(4):351-8), either alone, or in combination withother drugs in special carries like chitosan hydrogels.

Also hyaluronic acid as an active agent has been suggested as beinguseful in the treatment of skin ulcers (U.S. Pat. No. 5,897,880). Inaddition, topically applied fibronectin (glycoprotein found in bloodplasma) has been reported as being useful for increasing the rate ofwound healing in corneal wounds (Nishida, Larch Ophthalmology, 101: 1046(1983)) and leg ulcers (Wysocki et al., Arch. Dermatol, 124: 175(1988)).

Indeed these and other substances (like transforming growth factors(TGF-[alpha] and TGF-beta) (Science, 233:532, 1986); insulin-like growthfactors (IGF-I and II); adhesion factors, such as fibronectin, lamininand vitronectin (Ann. Rev. Biochem, 52:961, 1983), chemical substances,such as retinoids and analogous compounds thereof (Am. J. Ophthalmol.,95, 353-358, 1983; Ann. Ophthal, 19, 175-180, 1987)) have been suggestedto positively influence wound healing under the circumstances studied.

EP 0575484 discloses a pharmaceutical composition for the regenerationand repair of mammalian tissues, which includes PDGF and dexamethasone.U.S. Pat. No. 5,183,805 discloses a pharmaceutical composition havingthe effect of regenerating tissues, which includes EGF.

Although such substances provide patients with partial wound relief,they need long healing time and fail to exhibit the optimum response totreatment. Moreover, substances like EGF are difficult to prepare, areunstable (J Pharmacobiodyn (1991) 14: 47-52) and might quickly beinactivated when applied to a wound.

Moreover, wounds remain serious clinical problems to be solved, andalternatives useful in promoting wound healing are required, forexample, alternatives that function via new and/or additionalmechanisms, thereby allowing for better treatment options.

It is therefore one of the objects of the current invention to providefor a substance useful in promoting wound healing or wound closure andin the treatment of a wide variety of wounds, either alone or incombination with other substances known in the art, and that is easy toprepare in large amounts and is very stable.

Wounds typically occur on the skin. The skin is a vital organ ensuringmultiple functions such as sensitive functions, protective functionsfrom external aggressions, as well as immunological, metabolic orthermoregulatory functions. These roles are made possible due to acomplex structure that associates various tissues. The skin consists ofthree superimposed distinct layers: epidermis, dermis and hypodermis.The epidermis is a coating epithelium, which constitutes the externalstructure of the skin and provides its function of protection. Thisfunction is provided by the cohesion of the epithelial cells and by theproduction of a filamentous and resistant protein, keratin.

Today, the cosmetic industry seeks active ingredients, which are notonly able to protect and maintain skin but also active ingredients,which are able to improve its appearance as well as the well-being ofthe individuals who use it. A further purpose of the invention is tooffer new substances and use thereof, which have preventive and curativeaction on the skin in such phenomena like manifestations of aging,damage to skin tissue, like wounds, and the like.

It is therefore an object of the current invention to provide for asubstance that is useful in promoting wound healing and/or woundclosure, that can be used in the treatment of a wide variety of wounds,or the treatment of the skin (damage of the skin) either alone or incombination with other substances known in the art, and that is easy toprepare in large amounts and is very stable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a typical in vitro scratch wound closure experimentin which parotid saliva or a peptide according to the invention, after16 hours, induces wound closure of an epithelial monolayer. Differentsalivary secretions, 30% (v/v) in SFM, were tested in this model withsaliva buffer as a negative control. FIG. 1B is a graph showing parotidsaliva was the only secretion exhibiting induced wound closure. Even,the other secretions inhibited wound closure. Human whole stimulatedsaliva mainly consists of parotid saliva. FIG. 10 shows the results ofwound closure and EGF concentrations of six different persons. All butone person showed significant induced wound closure. Furthermore, EGFconcentrations do not correspond to the degree of wound closure,indicating that different factor(s) are responsible. FIG. 1D is a graphshowing that, to fully rebut EGF's role as wound closure inducing factorin human parotid saliva, the activation of its receptor was inhibited.With the receptor blocked the induction by parotid saliva not onlyremained, but even was boosted. This indicates responsibility of thisreceptor for the closure in the control and thus the background in ourassay, most likely caused by endogenous production of ligands for theEGF receptor. Of course, EGF, at a concentration of 5 ng/ml, was able toinduce wound closure with rates comparable to that of parotid saliva(FIG. 1D). Addition of AG1478 however, abolished the effect as expected.

FIG. 2 is a graph showing that the EGFR independent induced woundclosure of saliva as shown in FIG. 1 is affirmed by doing the same forHistatin 2. Also confirmed are the previously mentioned studies, whichshow EGF induced wound closure is p38^(MAPK) dependent. Histatin 2induced wound closure however, is not p38^(MAPK) but appears ERK1/2dependent.

FIG. 3 is a graph showing the results of various amino acids and/orpeptides (SEQ ID NOs: 5, 12, 11, 10, 9, 8, 26, 27, 31, 5, 19, 17, 15,13, 21, 5, 20, 23, 24, 18, 32, 25, 16, 28, 29, and 33 in descendingorder) according to the invention in wound closure experiments.

FIG. 4 is a graph showing the relative wound closure activity of eitherthe cyclic peptide (C-Hst1) or the linear peptide (Hst1) at differentconcentrations of each of the peptides.

DETAILED DESCRIPTION

It has surprisingly been found that the above-mentioned objects can beachieved by providing for the use of a peptide comprising

-   -   a. at least one amino acid sequence of at least 6, preferably at        least 7, most preferably at least 8 amino acids adjacently        present in Histatin 1, 2 and/or 3; and/or    -   b. at least one amino acid sequence having substitution,        deletion and/or insertion of at most 3, preferably at most 2,        most preferably at most 1 amino acid(s) in said amino acid        sequence of at least 6, preferably at least 7, most preferably        at least 8 amino acids adjacently present in Histatin 1, 2        and/or 3,        -   in the preparation of a medicament for the regeneration of            tissue and/or for the treatment of skin and/or for the            treatment of a wound.

Preferably, the use of the peptide according to the invention is for thetreatment of skin, more preferably for the treatment of a wound (eitherpresent on the skin or not).

Preferably the at least one amino acid sequence of at least 6,preferably at least 7, most preferably at least 8 amino acids isadjacently present in Histatin 1 and/or 2.

Even more preferably, as can be witnessed from the Examples, the aminoacid sequence comprised in the peptide according to the invention, andadjacently present in Histatin 1, 2 and/or 3 is at least 10, 11, 12, 13,14, or 15 amino acids long.

The term “peptide” is known in the art and relates to any compoundconsisting of two or more amino acids, joined by a peptide bond.Peptides can comprise of several amino acids, for example 10, 20, 50 or100, but within the context of the current invention peptides are notlimited to such numbers, but also include such larger peptides likepolypeptides or proteins. However, smaller peptides are preferred, asdiscussed below.

The term “an amino acid sequence having substitution, deletion and/orinsertion of at most 3, preferably at most 2, more preferably at most 1amino acid” is known to the person skilled in the art. With“substitution” is meant within the context of the current invention, thereplacement of an amino acid (for example in the amino acid sequenceaccording to any of SEQ. ID. NO. 1.-SEQ. ID. NO. 29) with another. Withthe term “deletion” is meant the removal of an amino acid (for examplefrom the amino acid sequence according to any of SEQ. ID. NO. 1.-SEQ.ID. NO. 29). With the term “insertion” is meant the introduction of anamino acid at any position within the amino acid sequence (for examplein any of the amino acid sequences according to SEQ. ID. NO.1-SEQ. ID.NO. 29).

Within the context of the current invention, in the case of asubstitution, deletion or insertion, at most 3, preferably at most 2,more preferably at most 1 amino acid are/is substituted, deleted and/orinserted. Any substitution, deletion or insertion is allowable withinthe context of the current invention as long as the obtained peptideshows wound closure activity as can be determined as described in theexamples (by comparison to a control).

The term “regeneration of tissue” is known to the person skilled in theart and relates to the repair, replacement, functional recovery andultimate regeneration of damaged tissues, either in a living body(including skin), or outside the body.

Within the current invention, the peptide according to the invention canthus be used in a medicament that can be applied to a living body, forexample a human or an animal, but can also be used to, outside the body,regenerate tissue, for example in the preparation of skin-grafts and thelike. Once developed, such tissues are transplanted into patients toinitiate the repair and rejuvenation process.

Preferably, however, the peptide is used in the preparation of amedicament for the treatment of and on a living body, for theregeneration of tissue, for the treatment of skin or eyes, or,preferably for the treatment of a wound.

The term “skin” is known to the person skilled in the art and within thecontext of the current invention has its normal meaning.

The term “wound” is known to the person skilled in the art and relatesto damaged tissues. A wound is a type of physical trauma where theintegrity of the skin or tissue is disrupted as a result from i.e.external force, bad health status, aging, exposure to sunlight, heat orchemical reaction or as a result from damage by internal physiologicalprocesses. If the outer layer of a tissue is damaged the wound isconsidered an open wound.

Wound closure is the process of regenerating the covering cell layers ofa tissue. Promoting wound closure means creating a positive effect inthe regeneration of the covering cell layers. The positive effect can bean acceleration of the process or a decrease of the damaged area of thewound.

Non-limitative examples of wounds are:

-   -   A burn wound is the injury resulting from exposure to heat,        electricity, radiation (for example, sunburn and laser surgery),        or caustic chemicals.    -   Ulcers    -   Wounds in Diabetes Mellitus are typically foot injuries due to        numbness caused by nerve damage (diabetic neuropathy) and low        blood flow to the legs and feet. The most serious injury is a        foot ulcer. Diabetic foot ulcers are at very high risk of        becoming infected, and sometimes they cannot be healed.        Non-healing foot ulcers are a frequent cause of amputation in        people with diabetes.    -   Decubitus wounds, decubitus (bedsores), i.e. lesions caused by        unrelieved pressure to any part of the body, especially portions        over bony or cartilaginous areas.    -   Wounds due to external force damaging the tissue    -   Skin wounds due to aging or the environment. This includes for        example splits, dry skin, roughness of the skin and the like.

During research it was surprisingly found that when peptides comprisingof at least one amino acid sequence of at least 6, preferably at least7, most preferably at least 8 amino acids adjacently present in Histatin1, 2 and/or 3; and/or at least one amino acid sequence havingsubstitution, deletion and/or insertion of at most 3, preferably at most2, most preferably at most 1 amino acid(s) in said amino acid sequenceof at least 6, preferably at least 7, most preferably at least 8 aminoacids adjacently present in Histatin 1,2 and/or 3, were applied toexperiments to determine wound-closure (tissueregeneration/proliferation, repair of the skin), effective wound closure(repair of the skin, regeneration) was observed (see Examples).

Therefore such peptides comprising such amino acid sequences and showingeffective wound closure, as can be determined as described in themethods, are very useful in the preparation of a medicament for theregeneration of tissue and/or for the treatment of skin and/or for thetreatment of a wound.

Without being bound by theory it is believed that a peptide comprisingan amino acid sequence according to the invention interacts with variouscells involved in the skin or wound healing process, possibly byinteracting with receptors present on such cells. Because of suchinteractions, it is believed that cell-proliferation (tissueregeneration) is induced, leading to efficient closure of for example awound or repair of the skin. Results suggest that in contrast to EGFinduced wound closure, which is p38^(MAPK) dependent, the peptidesaccording to the invention do not act via such p38^(MAPK) pathway, butappears to be ERK1/2 dependent.

Histatins 1,2 and 3 are known in the art and belong to a group ofhistidine-rich antimicrobial peptides, found in the saliva of man andsome higher primates.

There are 12 members of this family known and they are the products of 2different genes, Htn 1 and Htn 2. Histatin 1 and 2 are the products ofHtn 1 and Histatin 3 is a product of Htn 2. Histatin 1, 2 and 3 arelinear peptides that are relatively easy to produce and are therefore animprovement over growth factors for use in stimulating wound healing.

Histatins are known for their antifungal properties. It has beenestablished that histatins bind to a receptor on the fungal cellmembrane and enter the cytoplasm where they target the mitochondrion.They induce the non-lytic loss of ATP from actively respiring cells,which can induce cell death.

In addition, they have been shown to disrupt the cell cycle and lead tothe generation of reactive oxygen species. Their mode of action isdistinct from those exhibited by the conventional azole and polyenedrugs.

Indeed, the possibility of utilizing histatins for targeting fungalinfections of the oral cavity is being actively pursued with theantifungal properties of topical histatin preparations andhistatin-impregnated denture acrylic being evaluated. Initial clinicalstudies are encouraging, having demonstrated the safety and efficacy ofhistatin preparations in blocking the adherence of the yeast Candidaalbicans to denture acrylic, retarding plaque formation and reducing theseverity of gingivitis (Reviewed by Kevin Kavanagh, Susan Dowd; Journalof Pharmacy and Pharmacology Vol. 56, No. 3, pages 285 (2004).)

Interestingly, it has been found that the peptides and/or amino acidsequences according to the invention, including Histatin 1, Histatin 2and Histatin 3, show very advantageous effects with respect to tissueregeneration, skin repair and/or wound closure. In strong contrast, andcompletely unexpected, Histatin 5 showed no effect with respect totissue regeneration and/or wound closure (see Examples). Histatin 5 istherefore not included in the peptides and/or amino acids according tothe current invention.

It is therefore clear that the currently found activity is for examplenot attributable to the generally described antifungal activity ofHistatins towards for example Candida albicans, as firstly Candidaalbicans was not present in the methods for determining the effect ofHistatins on regeneration of tissue and/or wound closure, secondlybecause there is no relationship between the antifungal activity of thepeptides and the activity with respect to regeneration of tissue and/orwound closure, and thirdly Histatin 5, being the most active peptidetowards Candida albicans has no activity towards regeneration of tissueand/or wound closure (see Examples enclosed herein).

Therefore, the peptides disclosed in for example JP06287146, includingHistatin 5, are clearly distinct from the peptides according to thecurrent invention, both in structure as well as in the mechanisms bywhich they might be relevant with respect to tissue regeneration, skinrepair and/or wound closure. In particular, in the case the peptideaccording to the invention comprises an amino acid sequence according toSEQ.ID. NO.3, it is preferred it is with the proviso that such peptideis not a peptide as specifically disclosed in JP06287146.

However, in a preferred embodiment, such peptides might advantageouslybe combined with the peptides according to the current invention.

In a preferred embodiment there is provided for the use of a peptidecomprising of at least one amino acid sequence of at least 6, preferablyat least 7, most preferably at least 8 amino acids adjacently present inHistatin 1, 2 and/or 3 wherein said amino acid sequence is selected fromthe group consisting of SEQ. ID. NO. 1, SEQ.ID. NO. 2 and SEQ.ID.NO.3,or at least one amino acid sequence having substitution, deletion and/orinsertion of at most 3, preferably at most 2, most preferably at most 1amino acid(s) in said amino acid sequence selected from the groupconsisting of SEQ. ID. NO. 1, SEQ.ID. NO. 2 and SEQ.ID.NO.3.

In an even more preferred embodiment there is provided for the use of apeptide comprising of at least one amino acid sequence of at least 6,preferably at least 7, most preferably at least 8 amino acids adjacentlypresent in Histatin 1, 2 and/or 3 wherein said amino acid sequence isaccording to SEQ.ID. NO. 2, or at least one amino acid sequence havingsubstitution, deletion and/or insertion of at most 3, preferably at most2, most preferably at most 1 amino acid(s) in said amino acid sequenceaccording to SEQ.ID. NO. 2.

During research, in an attempt to establish the minimal requirements ofthe amino acid adjacently present (meaning next to each other in desequence) in Histatin 1,2 and/or 3, it was surprisingly found that whena peptide comprising an amino acid sequence according to SEQ. ID. NO. 1,SEQ.ID. NO. 2 and/or SEQ.ID.NO.3, and/or an amino acid sequence havingsubstitution, deletion and/or insertion of at most 3, preferably at most2, more preferably at most 1 amino acid in the amino acid sequenceaccording to SEQ. ID. NO. 1, SEQ.ID. NO. 2 and/or SEQ.ID.NO.3, wasapplied to experiments to determine wound-closure (tissueregeneration/proliferation, repair of the skin), effective wound closure(repair of the skin, regeneration) was observed (see Examples).

In other words, the peptides according to the invention, comprising ofor consisting of an amino acid sequence according to SEQ. ID. NO. 1,SEQ.ID. NO. 2 and/or SEQ.ID.NO.3 (or an amino acid sequence havingsubstitution, deletion or insertion of at most 3, preferably at most 2,more preferably at most 1 amino acid in the amino acid sequenceaccording to SEQ. ID. NO. 1, SEQ.ID. NO. 2 and/or SEQ.ID.NO.3), can showefficient activity towards wound-closure (skin repair, tissueregeneration/cell-proliferation) by a mechanism that is independent fromthe known wound-closure factor EGF. Thereby treatment of such wounds ortissue damage can be now be further improved by the provision of analternative (or additional) mechanism that can be targeted.

Therefore also claimed is the receptor with which the peptides accordingto the invention, having an amino acid sequence according to theinvention, interact and thereby induce wound healing; use of suchreceptor in the detection of compounds that might be useful in theregeneration of tissue, in particular in the treatment of skin orwounds; and use of such compounds in medicaments and the like in tissueregeneration, preferably for and in the treatment of a wound or thetreatment of skin.

In another preferred embodiment, there is provided for the use of apeptide comprising an amino acid sequence selected from any of the aminoacid sequences according to SEQ. ID NO. 4-SEQ ID. NO. 29, or an aminoacid sequence having substitution, deletion and/or insertion of at most3, preferably at most 2, more preferably at most 1 amino acid in theamino acid sequence according to SEQ. ID. No. 4-SEQ ID. NO 29,preferably said substitution, deletion and/or insertion not being in anamino acids sequence according to SEQ.ID.NO. 1, SEQ. ID. NO. 2, and/orSEQ. ID. NO. 3.

During experiments performed by the inventors it was observed that inaddition to the above disclosed amino acid sequences, peptides having anamino acids sequence according to SEQ. ID. NO. 4-SEQ ID. NO. 29 showbeneficial effects with respect to regeneration of tissue, wound closureand or treatment of the skin.

As can be seen in the examples, the peptides or amino acid sequenceshave been modified by the removal of 2, 4, 6, 8, 12 etc amino acids incomparison to for example the amino acid sequence SEQ. ID. NO 5(Histatin 2). Moreover, all these sequences showed beneficial effects inthe tests performed. It will thus be appreciated by the skilled personthat in addition to those amino acids sequences according to SEQ. ID. NO4-SEQ. ID. NO. 29, in addition also the amino acids wherein1,3,5,7,9,11, 13, etc. amino acids have been removed in comparison toSEQ. ID. NO. 5, for example by removal of the tripeptide RKF, thepentapeptide RKFHE, etc. from the N-terminus of SEQ. ID. NO. 5, or bythe removal of the tripeptide YDN, or the pentapeptide YLYDN, etc. fromthe C-terminus of SEQ. ID. NO. 5, are also included as amino acids thatcan be comprised (or form) the peptides according to the invention.Obviously, the removal of amino acids can also be from both theN-terminus and the C-terminus, as long as the remaining peptide showsactivity with regard to wound closure, for example as determined in theExamples.

In another preferred embodiment, the peptide according to the inventioncomprises or preferably consists of an amino acid sequence selected fromthe group consisting of SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6,or parts or fragments thereof and which show wound closure activity.

The peptide according to SEQ. ID NO. 4 is also known as Histatin 1; thepeptide according to SEQ. ID NO. 5 is also known as Histatin 2; thepeptide according to SEQ. ID NO 6 is also known as Histatin 3.

As will be appreciated, and based upon the current disclosure, theskilled person will without any further inventive skill be capable ofdetermining the wound closure activity of fragments or parts of aminoacid sequences selected from the group consisting of SEQ. ID. NO. 4,SEQ. ID. NO. 5, and/or SEQ. ID. NO. 6, for example as is described indetail in the Examples. It will be understood that such parts orfragments are part of the current invention. Preferably, said parts orfragments are derived from the amino acid sequence according to SEQ.ID.NO. 5.

As will be understood by the skilled person, the amino acids and thepeptide comprising such amino acid sequences as disclosed in the currentinvention include those in which at least one functional grouping (inparticular the amine and carboxylic groupings) are protected with aprotective grouping. As the peptide according to the invention is to beapplied to tissue, skin or wound, it is beneficial, for resistance todegradation, to use a protected form of the peptide. The form ofprotection must obviously be a biologically compatible form and must becompatible with cosmetic use or the field of pharmaceuticals. Manybiologically compatible forms of protection can be considered, they arewell known to the person skilled in the art, such as for example theacylation or the acetylation of the amino-terminal, cyclization or theamidation or the esterfication of the carboxy-terminal. Thus, theinvention also concerns a use such as previously defined andcharacterized by the fact that the peptide is in a protected form.

Peptides, objects of this patent, can be obtained either by traditionalchemical synthesis (for example as described in the Examples, or insolid phase or in homogeneous liquid phase), or by enzymatic synthesis(Kullman et al., J. Biol. Chem. 1980, 225, 8234) from constitutive aminoacids or from their derivatives. Peptides relating to the invention canalso be obtained by fermentation of a strain of bacteria, modified ornot, by genetic engineering to produce peptides of the sequence, aspreviously indicated, and their fragments.

Also, there is provided for nucleotides (DNA, cDNA, RNA, etc.) encodingfor a peptide comprising an amino acid sequence according to theinvention, and use thereof for providing for the peptides according tothe invention.

In another embodiment, the peptide comprises or consists of at least 8,more preferably at least 10, even more preferably at least 20, mostpreferably at least 27 amino acids.

It has been found that if the peptide according to the inventioncomprises or consists of at least 8, more preferably at least 10, evenmore preferably at least 20, most preferably at least 27 amino acids,there is provided for a peptide, which shows good activity with regardto tissue regeneration, skin treatment and/or wound closure.

It is believed that, in addition to for example the amino acid sequenceaccording to SEQ. ID. NO 1-SEQ.ID.NO. 29, or an amino acid sequencehaving substitution, deletion or insertion of at most 3, preferably atmost 2, more preferably at most 1 amino acid in the amino acid sequenceaccording to SEQ. ID. NO 1-SEQ.ID.NO. 29, the additional amino acidscontribute positively to the interaction of the peptide with the cellsinvolved or present in tissue regeneration, skin repair and/or woundclosure.

It will therefore be understood by the person skilled in the art thatany amount of additional amino acids (next to the amino acids sequencesaccording to the invention) might be present in the peptide according tothe invention as long as this does not substantially negativelyinfluence the activity of the peptide in closing a wound, for example asdetermined in the methods as described in the examples.

In another preferred embodiment there is provided that the peptide foruse in the current invention comprises 8-40 amino acids, more preferably12-39 amino acids, even more preferably 27-38 amino acids.

As has been discussed above, it was found that in particular peptidescomprising 8-40 amino acids, more preferably 12-39 amino acids, evenmore preferably 27-38 amino acids are in particular efficient in theregeneration of tissue, in skin treatment and in particular in closing awound, as described in the examples.

It is believed that, in addition to for example the amino acid sequenceaccording to SEQ. ID. NO 1-29, or an amino acid sequence havingsubstitution, deletion or insertion of at most 3, preferably at most 2,more preferably at most 1 amino acid in the amino acid sequenceaccording to SEQ. ID. NO 1-29, the additional amino acids contributepositively to the interaction of the peptide with the cells involved orpresent in tissue regeneration, skin repair and/or wound closure.

Although, as mentioned above, the peptides my comprise of more aminoacids than described in the above ranges of amino acids, it is howeverbelieved that when the peptide comprises of too many amino acids,interaction of the peptide according to the invention with cells andreceptors might be negatively influenced, thereby reducing the efficacyof the peptide according to the invention in tissue regeneration, skintreatment and/or treatment of a wound (wound closure). Therefore, it ispreferred that the peptide according to the invention comprises at mostno more than (about) 100 amino acids.

It will therefore be understood by the person skilled in the art thatany amount of additional amino acids might be present in the peptideaccording to the invention as long as this does not substantiallynegatively influence the activity of the peptide in closing a wound (inregeneration of tissue or in treatment of skin), for example in themethods as described in the examples.

In another preferred embodiment there is provided that the peptideaccording to the invention is an L-peptide, or in other words, thepeptide according to the invention is made up of L-amino acids.

Indeed, with the term “L-peptide” is meant a peptide wherein all aminoacids are in the L-form, i.e. in the form as they are normally producedin a living body.

Nineteen of the essential twenty amino acids have the property of“chirality” or handedness. The only achiral essential amino acid isglycine. To describe a chiral compound, the prefixes D and L are used torefer to the configuration of the molecule around its chiral centre. Thechiral centre of an amino acid is the alpha carbon, and whether an aminoacid is of the D configuration or the L configuration depends upon thestereoisomeric conventions established by Emil Fisher. A chiral aminoacid can exist as stereoisomers, which are identical chemical structuresthat are mirror images of each other. Both stereoisomers are oftenreferred to as an enantiomeric pair, and a stereoisomer is oftenreferred to as an enantiomer, which is a nonsuperimposable mirror imageof the other stereoisomer/enantiomer.

All of the naturally occurring chiral amino acids exist in the Lconfiguration, and are referred to generally as L-amino acids. Thestereoisomer of each chiral amino acid in the L-configuration isreferred to as a D-amino acid.

It was found that in case D-amino acids were used instead of L-aminoacids in the peptide according to the invention, activity of the peptideaccording to the invention in tissue regeneration and/or wound closurewas significantly reduced in comparison to when the natural occurringL-amino acids made up a peptide according to the invention.

It is believed that a peptide comprising of only L-amino acids providesfor better interaction with cells and or receptors involved in tissueregeneration and/or wound closure.

In another embodiment, there is provided for the use according to theinvention wherein the peptide is a cyclic peptide.

It has been found that when, for example, a peptide comprising an aminoacid sequence according to SEQ. ID. NO. 4 was provided in cyclic form,the activity of such cyclic form of the peptide is dramaticallyincreased in comparison to the linear form. In other words, in order toachieve the same skin repair effect, wound healing or wound closureeffect, or the same effect with regard to tissue regeneration, as forexample can be determined with the methods described in the examples, a(much) lower concentration of a cyclic peptide according to theinvention can be used in comparison to the same peptide in linear form.Alternatively, when using the same concentration of a linear form of apeptide according to the invention of a cyclic form of a peptideaccording to the invention, dramatically increased skin repair, woundclosure, tissue regeneration can be established with the cyclic peptidein comparison to the linear form of the same peptide (and as for examplegiven in any of SEQ.ID.NO.1-29).

In other words, in a preferred embodiment there is provided for apeptide according to the invention in cyclic form, more in particularthe cyclic peptide comprises an amino acid sequence according to any ofSEQ.ID. NO. 1-SEQ.ID NO. 29.

The person skilled in the art knows how to provide for a cyclic peptide,and many suitable methods have been described in the art. For example,U.S. Pat. No. 6,555,650 discloses a method for providing cyclic analogsof histatins having substantial homology to His 5 (the method forpreparing such cyclic forms can be used within the context of thecurrent invention).

In addition, Goncalves et. al. (Tetrahedron 61 (2005) 7789-7795)describes a method that can be applied for forming cyclic peptidesaccording to the current invention. In short, in such method a linearpeptide is constructed using standard Fmoc chemistry and on-resincyclization was enabled after selective deprotection of the C-terminalgroup with hydrazine/DMF.

It is believed that the improved characteristics of such cyclic peptidesin comparison to the linear peptides, might be due to increasedmetabolic stability, potency, receptor selectivity and/orbioavailability. It will therefore be understood by the skilled personthat the cyclic peptides are not limited to the cyclic peptide as shownin the examples, but include any cyclic peptide comprising an amino acidsequence selected from any of the amino acid sequences according to SEQ.ID NO. 1-SEQ ID. NO. 29, or an amino acid sequence having substitution,deletion and/or insertion of at most 3, preferably at most 2, morepreferably at most 1 amino acid in the amino acid sequence according toSEQ. ID NO. 1-SEQ ID. NO. 29, preferably said substitution, deletionand/or insertion not being in the amino acids sequence according to SEQ.ID. NO. 1, SEQ. ID. NO. 2, and/or SEQ. ID. NO. 3.

Also included are those cyclic peptides comprising an amino acidsequence according to SEQ. ID. NO. 1, SEQ. ID. NO. 2, and/or SEQ. ID.NO. 3—or an amino acid sequence having substitution, deletion and/orinsertion of at most 3, preferably at most 2, more preferably at most 1amino acid in the amino acid sequence according to SEQ. ID. NO. 1, SEQ.ID. NO. 2, and/or SEQ. ID. NO. 3.

In addition, as explained above, also included are those cyclic peptidescomprising an amino acid sequence wherein 1,3,5,7,9,11, 13, etc. aminoacids have been removed in comparison to SEQ. ID. NO. 5, for example byremoval of the tripeptide RKF, the pentapetide RKFHE, etc. from theN-terminus of SEQ. ID. NO. 5, or by the removal of the tripeptide YDN,or the pentapeptide YLYDN, etc. from the C-terminus of SEQ. ID. NO. 5,or by removal from both the N-terminus and the C-terminus of SEQ. ID.NO. 5. These amino acids sequences can be comprised in a cyclic peptideaccording to the invention.

Therefore, in a further embodiment of the invention there is providedfor the use of a cyclic peptide according to the invention wherein thecyclic peptide comprises an amino acid sequence selected from any of theamino acid sequences according to SEQ. ID NO. 1-SEQ ID. No. 29, or anamino acid sequence having substitution, deletion and/or insertion of atmost 3, preferably at most 2, more preferably at most 1 amino acid inthe amino acid sequence according to SEQ. ID. NO. 1-SEQ ID. NO 29,preferably said substitution, deletion and/or insertion not being in theamino acids sequence according to SEQ. ID. NO. 1, SEQ. ID. NO. 2, and/orSEQ. ID. NO. 3

Indeed it has been found that when a cyclic peptide comprising an aminoacid sequence according to the invention is compared to the same peptidebut in linear form, the concentration of the cyclic peptide might be 5,even 10, even 100 times lower in comparison to the linear form of thesame peptide (for example a linear form of the amino acid sequenceaccording to SEQ.ID.NO. 4 (Histatin 1)).

Therefore, there is provided for use according to the invention of acyclic peptide according to the invention wherein said cyclic peptidehas a relative wound closure activity that is equal to the relativewound closure activity of a linear peptide according to SEQ. ID. NO. 4(HIS1) at a concentration of 1 μM (in the method according to theExamples), at a concentration of the said cyclic peptide that is atleast 5, more preferably at least 10, even more preferably at least 100times lower. In particular the said relative wound closure activity ismeasured according to the method of Example 1.

Any skilled person is capable of determining the relative wound closureactivity using the methods as described in the examples and can thuseasily determine the activity of any linear peptide of cyclic peptideaccording to the invention and compare such results.

In another embodiment of the invention, the peptide according to theinvention is a dimer or mulitmer, each monomer forming the dimer ormultimer preferably consisting of (being formed by) an amino acidsequence as defined above, even more preferably each monomer consistingof an amino acid sequence according to any of SEQ. ID. NO. 1-SEQ ID. NO.29.

The skilled person understand what the term “dimer” or “multimer” withinthe context of the current invention is intended to indicate, i.e., adistinct part of the peptide according to the invention in combinationwith another distinct part, either directly linked in a peptide bond orindirectly via a linking sequence or a cross-linker. The parts can belinked either in a parallel fashion (X₁-X₂-X₃- . . . Xn-Y-X₁-X₂-X₃- . .. X_(n)) , or, and thus included in the current invention, in anantiparallel fashion (X₁-X₂-X₃- . . . X_(n)-Y-X_(n)-X_(n−1)-X_(n−2)- . .. X_(1,)) in which X_(n) indicates the amino acid residue at position 1(starting from the N-terminus) and Y denotes the cross-linking betweenthe two “monomers”. In other words, a peptide according to the inventionmay comprise any fusion of at least two amino acid sequences accordingto the invention, each amino acid sequence according to the inventionforming a “monomer” within the context of the invention.

Further, and in addition to the example described above, such peptidemay comprise additional amino acids, preferably being according to anamino acid according to the invention, forming a further monomer withinthe context of the invention.

Preferably, the peptide is a dimer comprising two identical monomersaccording to the invention, and as described above, for example bothmonomers within the context of the invention having an amino acidsequence according to SEQ. ID. NO. 1-SEQ ID. NO. 29.

As will be understood by the skilled person, also included in thecurrent invention are those peptides according to the invention that areconjugated. Within the current invention “conjugation” is to beconstrued as the binding of a peptide according to the invention toanother material, in particular to a carrier, for example a polystyrenebead, BSA, an antibody or a delivery vehicle like. Thus, in anotherembodiment there is provided a peptide according to the invention thatis conjugated.

With respect to the observed increase in activity of the above-discussedcyclic form of a peptide according to the invention, it is believed thismight at least partially be due to the constraining of theconformational freedom of the linear peptides by the cyclization. Itwill therefore be understood by the skilled person that also comprisedare peptides according to the invention that have been constrained intheir conformational freedom, in addition to cyclic peptides. The personskilled in the art is aware of methods constraining the conformationalfreedom of peptides according to the invention, for example a methodwherein said peptide has been constrained in its conformational freedomby cross-linking or by the formation of sulphur-sulphur bridges. Forexample by the CLIPS technology as e.g. described by Lesauteur et al(JBC, 270, 6564-6569). This is achieved by substitution of non-criticalresidues with cysteine or by insertion of cysteine residues innon-critical positions of the molecule. Subsequently cyclization can beconducted by oxidative dimerization (by flushing with oxygen or air) orby treatment with diiodomethane, or by reaction with fast small aromaticscaffolds like α,α′-dibromo-m-xylene α,α′-dibromo-m-xylene.

In another preferred embodiment, in addition to the peptide according tothe invention, further a growth factor, preferably selected from thegroup consisting of platelet derived growth factor (PDGF), insulinlike-growth factor (IGF), transforming growth factor (TGF), hepatocytegrowth factor (HGF), epidermal growth factor (EGF), fibroblast growthfactor (FGF), is used in the preparation of the medicament, or in thetreatment of skin or wound.

As already discussed above, the peptides according to the inventioncontribute to tissue regeneration and/or wound closure and/or skintreatment by a mechanism different from that of for example EGF. It istherefore with advantage to combine compounds/drugs and the like withthe peptides according to the invention and that contribute toregeneration of tissue and/or closure of the wound (wound treatment)and/or skin treatment by mechanisms that are, preferably, independentfrom the mechanism by which the peptides according to the invention act.By combining said different mechanisms in one treatment, better resultsare obtainable.

In another preferred embodiment, there is provided for the use accordingto any of the previous clauses wherein the treatment of the skin is forthe treatment or prevention of aging of the skin, cellulites, dry skin,splits, wounds or wherein the wound is an internal wound, an oral wound,a skin wound, an external wound, an ulcer, and/or a decubitus wound,damage to the eye, a wound in the eye, for example conjunctiva. Otherconditions that can be treated with the peptides according to theinvention include oral ulcers, oral aphthous lesions, Burning mouthsyndrome, Burning tongue, psoriasis, eczema, and hair loss.

These types of skin damages and wounds are known to the person skilledin the art. An internal wound is a wound present in the body, forexample due to a surgical incision. An oral wound is a wound present inthe oral cavity. A skin wound is a wound present in the skin. Anexternal wound is to be understood as a wound that is visible andaccessible from outside the body. An ulcer is a lesion on the surface ofthe skin or a mucous surface. A decubitus wound is a wound or ulcerationcaused by prolonged pressure on the skin and tissues when one stay inone position for a long period of time, such as lying in bed. The bonyareas of the body are the most frequently affected sites, which becomeischemic under sustained and constant pressure. Aging of the skin is thechange in appearance of the skin due to time or exposure to theenvironment or the health status of an individual. Cellulites is thedefinition used in cosmetics relating to a wobbly or dimpled appearanceof the skin (orange skin in Dutch).

In a more preferred embodiment of the invention, the method is used forthe treatment of a skin wound. Skin wounds can be wounds in theepidermis or dermis of the skin. There are several types of wounds inwhich the skin or tissue may be in need of repair: abrasions,lacerations, incisions, punctures and avulsions and burns. Use of apeptide according to the invention can improve the general health statusof the skin. In another preferred embodiment of the invention, themethod is used for the treatment of an oral wound. Oral wounds arewounds in any part of the oral cavity wherein the oral mucosa isdamaged. In another preferred embodiment of the invention, the method isused for the treatment of an internal wound. Internal wounds are woundswherein cell layers of endodermal or mesodermal origin are damaged.Examples are wounds in arteries or venes, peritoneum or pericardium.

In another preferred embodiment there is provided for the use of thepeptide according to the invention in the preparation of a medicamentand wherein the medicament has an ionic strength of at least 20 mM, morepreferably 50 mM, even more preferably 100 mM, most preferably at least120 mM, before of after application to a wound.

The term ionic strength is known to the person skilled in the art, andcan for example be determined according to PAC, 1996, 68, 977 (IUPAC;)It has surprisingly been found that in contrast to for example anyantifungal activity, activity with respect to tissue regeneration and/orwound closure is maintained at such levels of ionic strength.

For example, when the peptide according to the invention is applied to awound which excretes some exudate, it will be active with respect totissue regeneration and/or wound closure, whereas, due to the presenceof a too high an ionic strength (due to ions being present in theexudate), peptides like Histatin 5 will not be functioning as aantifungal at such concentrations (see Examples).

In another aspect of the current invention, there is provided acomposition comprising a peptide according to the invention, and asdescribed above, characterized in that the composition further comprisesat least one pharmaceutically acceptable excipient.

In a preferred embodiment, the excipient is pharmaceutically acceptablein the treatment of a wound, i.e. does not negatively interfere with thetreatment of the wound and/or the regeneration of tissue.

In addition, the composition according to the invention can furthercomprise a growth factor, preferably selected from the group consistingof platelet derived growth factor (PDGF), insulin like-growth factor(IGF), transforming growth factor (TGF), hepatocyte growth factor (HGF),epidermal growth factor (EGF), fibroblast growth factor (FGF), for thereasons discussed above.

The compositions according to the discussion can suitable be used in thetreatments as described above.

In another preferred embodiment, the composition is in the form of asolution, an ointment, a salve, a balsam, a tincture, an elixir, aplaster, a bandage, a dressing material, an alginate dressing, a topicalsolution, an infusion, or a surgical rinse solution.

Preferably the composition comprises the peptide according to theinvention in an amount of about 0.0001 to 500 mg of the peptide permilliliter of gram of the medicament.

In case of the medicament/composition being in an aqueous form, i.e. asolution or an ointment, the pH of the medicament is between pH 3.0 andpH 9.0. In another preferred embodiment, said composition has an ionicstrength of at least 20 mM, more preferably 50 mM, even more preferably100 mM, most preferably at least 120 mM, before of after application toa wound, and as discussed above.

In another aspect of the current invention there is provided for amethod for the treatment of skin or treatment of a wound comprising thestep of applying to said skin or wound an amino acid a peptide orcompositions as defined herein. The said method is very useful in thetreatment of wounds, skin, or in the regeneration of tissue, and asdescribed herein. Preferably the medicament comprising the peptideaccording to the invention comprises about 0.0001 to 500 mg of thepeptide per milliliter of gram of the medicament. In case of themedicament in an aqueous form, i.e. a solution or an ointment, the pH ofthe medicament is between pH 3.0 and pH 9.0.

In another aspect of the current invention there is provided for the useof the peptides according to the invention is the cosmetic (non-medical)treatment of the skin. It has been found that even if there is nomedical need for the treatment of the skin, application of a peptideaccording to the invention to for example aged skin, or skin beingsubject to environmental stress (i.e. air pollution) advantageouslyshows improvement in the structure and appearance of the skin.

The above mentioned peptide can be solubilised in one or severalcosmetically or pharmaceutically acceptable solvents such as water,ethanol, propanol or isopropanol, propylene glycol, butylene glycol,dipropylene glycol, ethoxylated or propoxylated diglycols, cyclicpolyols or any combinations of these solvents, or are solubilised in acosmetic or pharmaceutical vector such as liposomes or are adsorbed onpowdered organic polymers, mineral supports such as talc and bentonites,and more generally solubilised in, or fixed on, any cosmetically orpharmaceutically acceptable vectors. It is of course obvious that thepeptide according to the invention can be used alone or in partnershipwith at least another active agent, in or for the preparation of acosmetic and/or dermatological and/or pharmaceutical composition. Thepeptide according to the invention can favorably be used as skin careproduct and treatment agent for the skin. Skin care product andtreatment agent refer to agents which in a general way have a repairingand revitalizing activity allowing, inter alia, the skin and/orsuperficial body growths to better react to the aggressions which theycan be subjected to.

It will be understood that the peptide according to the invention can beused alone or in association with at least another active agent. In thecomposition according to the invention, the peptide can be a mixture ofpeptide derivatives and/or consisting of amino acids derivatives.

The peptides according to the invention can therefore suitable be usedin cosmetic preparation intended for application to the skin.

It will be understood by the person skilled in the art that in additionto amino acids, the peptide may further contain such commonly usedgroups like signal-peptides or chemical groups like protection groupslike tBoc, tags, of even be associated with or included in for examplenano-particles and liposomes, for example carrying an additional drug,for example useful in the treatment of wounds. Peptides that arechemically modified such as by glycosylation, pegylation, acetylation,methylation, ubiquitination, hydroxylation, palmitoylation,phosphorylation can also be used as long as these modifications do nothamper the wound closing properties and/or tissue regeneration and ortreatment of the skin.

In another aspect there is provided for an amino acid sequence, peptideor composition as defined in any of the previous claims, preferably apeptide comprising or consisting of an amino acid sequence according toany of SEQ. ID. NO. 1 -SEQ.ID. NO. 29, or an amino acid sequence havingsubstitution, deletion and/or insertion of at most 3, preferably at most2, more preferably at most 1 amino acid in the amino acid sequenceaccording to SEQ. ID. NO.1-SEQ ID. NO. 29.

Also provided is the amino acid sequence, peptide or compositionaccording to the invention for use as a medicament. In particularprovided are amino acid sequence, peptide or composition according tothe invention in the treatment of skin, wound or in tissue regeneration,preferably a peptide comprising or consisting of a amino acid sequenceaccording to any of SEQ. ID. NO. 1 -SEQ.ID. NO 29.

EXAMPLES Example 1

1. Experimental Conditions

1.1 Cell Culture

The human buccal epithelial cell line TR146 (Rupniak et al; J NatlCancer Inst. 1985 October; 75(4):621-35) was kindly provided by CancerResearch UK. Cells were grown in Dulbecco's modified Eagle medium with4.5 g/l glucose (Invitrogen), 10% FCS (HyClone) and 100 U/ml penicillin,100 mg/ml streptomycin, and 250 ng/ml amphotericinB (Antibioticantimyotic solution, Sigma,), at 37° C., 95% humidity and 5% CO2. Cellswere maintained until near confluence and then briefly treated with0.25% trypsin-EDTA 1× (Invitrogen) to detach them, counted in ahepacytometer and seeded into new flasks or multi-well plates at therequired cell density of about 1.5×10×5 cells/well.

1.2 Saliva Collection

Saliva was collected as previously described (Veerman et al., 1996 Eur JOral Sci. 104:346-52.). Human whole stimulated saliva (WSS) wascollected by chewing on a piece of parafilm. Parotid saliva collectedwith the aid of a Lashley cup positioned over Stensons duct into apolypropylene tube. The fluid accumulating in the mouth during thecollection of the parotid secretion (designated ex-parotid saliva),which is mainly composed of the combined secretions of the submandibularand sublingual glands, was collected simultaneously by spitting into anice-cooled vessel. Submandibular saliva (SM) was collected using acustom-made collection device placed in the floor of the mouth over theexits of ducts of the submandibular glands. Before use, all salivasamples were sterilized using a 0.45 μm pore rezist filter (Whatman).Saliva buffer was assembled based on the properties of stimulatedparotid saliva with pH=7.3 and consisted of 30 mM Na2CO3, 10 mM KCl, 6mM K2HPO4, 3 mM KSCN, 1 mM CaCl2 and 0.1 mM MgCl2. The high molecularweight salivary mucin MUC5B was isolated by ultracentrifugation and gelfiltration chromatography, as previously described (Veerman et al.,1991, Arch Oral Biol. 36:923-32.).

1.3 EGF Enzyme-Linked Immuno Sorbent Assay (ELISA)

EGF concentrations in saliva were measured by ELISA using human EGFcytoset™ kit, following manufacturer's instructions (Biosource).

1.4 In Vitro Wound Healing Experiments

Wound assays were performed as previously described (Matthay et al.,1993, J Cell Sci. 106 (Pt 3):869-78). In brief, cells were grown in12-well plates until confluence, and then were serum deprived for 24hours in keratinocyte serum free medium (SFM (Invitrogen)). In each wella scratch was made in the cellular monolayer using a sterile tip. Thewounded monolayer was washed with SFM to remove cellular debris, and thefollowing conditions were applied:

-   -   (i) Whole saliva and salivary secretions applied in 30% (v/v) in        SFM. As a negative control 30% (v/v) saliva buffer was used.    -   (ii) (ii) rhEGF (Invitrogen) as a positive control    -   (iii) rp-HPLC fractions containing salivary proteins;    -   (iv) synthetic peptides: the experiments with isolated proteins        and synthetic peptides were performed in full SFM.

The width of the wound area was determined microscopically immediatelyafter wound creation and at approximately 16 hours after wounding. Therelative closure was calculated by relating the closure to that of thenegative control.

For inhibitor studies; directly after wounding cells were exposed toinhibitors of ERK1/2 (U0126, 5 μM, LC Laboratories), EGFR (AG1478, 1 μM,Calbiochem), and p38 MAPK (SB203580, 5 μM; LC Laboratories). Experimentswere done in quadruplicate and analyzed using One-way ANOVA to determinesignificance with additional LSD post hoc test, P<0.05 was consideredsignificant.

1.5 HPLC Chromatography of Parotid Saliva and Identification of Proteins

Parotid saliva (2 ml) from one donor was subjected to reversed phasehigh-performance liquid chromatography (RP-HPLC) on a C8 column by usinga RP-HPLC system (Jasco). The fractions that exhibited wound closureproperties were pooled and reconstituted in 1 ml HPLC-grade water andsubjected to RP-HPLC chromatography on a C18 column. Resulting fractionsthat induced wound closure, were analyzed with ion trap massspectrometry with a LCQ Deca XP (Thermo Finnigan).

1.6 Peptide Synthesis

Synthetic peptides were synthesized using by solid phase peptidesynthesis using Fmoc chemistry with a MilliGen 9050 peptide synthesizer(Milligen-Biosearch), FITC labeling, purification and authenticityconfirmation as previously described (den Hertog et al., 2005, BiochemJ. 388:689-95).

1.7 Proliferation Assay

Approximately 1×10⁴ cells were seeded in 24 well plates and incubatedovernight. Cells were treated with Karyomax™ (Invitrogen) for 6 hours.After washing with SFM, they were incubated for 24 h and pulsed with 1μCi of [methyl-3H]-thymidine for 24 h at 37° C. Then cells were washedwith ice cold PBS, 100% methanol and 5% TCA. The acid-insoluble materialwas dissolved in 0.5 M NaOH at room temperature and the level ofradioactivity was counted using a liquid scintillation counter(Packard). Values were converted from absolute counts to a percentage ofthe control.

1.8 Microscopy and Imaging

Microscope: Leica DM IL PLAN 4-40× magnification, 0.5 lens; camera:Leica DFC320; and acquisition software: Leica IM500. Images werequantified using Adobe Photoshop CS3. Additionally, brightness/contrastlevels of the co-localization studies were altered with Adobe PhotoshopCS3. FITC labeled histatin was incubated with the cells for two hours.PI was added to check for membrane disruptive dependent internalization.100 mM Azide was used for 1 hour. Cells were trypsinized as describedabove. Co-localization studies were performed using Mitotracker(Invitrogen) and Lysotracker (Invitrogen) following manufacturer'sinstructions.

2. Results

2.1 There is no correlation between wound closure properties of salivaand different salivary secretions vs. saliva buffer and their EGFconcentrations. Since the majority of studies on wound healing dealtwith saliva from rodents, it was first affirmed that human salivarysecretions indeed accelerate wound closure in the model used herein.Some studies have found salivary substances enhancing migration andproliferation, whereas other studies found factors in saliva whichinhibited attachment and migration, such as MUC5B. Although, most ofthese studies use isolated proteins, which makes it difficult tointerpret its context.

FIG. 1A illustrates a typical in vitro scratch wound closure experimentin which parotid saliva, after 16 hours, induces wound closure of anepithelial monolayer. Different salivary secretions, 30% (v/v) in SFM,were tested in this model with saliva buffer as a negative control.Parotid saliva was the only secretion exhibiting induced wound closure(FIG. 1B).

Next, the role of EGF's in parotid induced wound closure was studied.Wound closure and EGF concentrations of six different persons wasstudied (FIG. 1C). All but one person showed significant induced woundclosure, and EGF concentrations does appear not to correspond to thedegree of wound closure, indicating that different factor(s) areinvolved. To fully rebut EGF's role as wound closure inducing factor inhuman parotid saliva we inhibited the activation of its receptor with 1μM AG1478 (FIG. 1D). With the receptor blocked the induction by parotidsaliva not only remained, but even was boosted. This indicatesresponsibility of this receptor for the closure in the control and thusthe background in our assay, most likely caused by endogenous productionof ligands for the EGF receptor. Of course, EGF, at a concentration of 5ng/ml, was able to induce wound closure with rates comparable to that ofparotid saliva (FIG. 1D). Addition of AG1478 however, abolished theeffect as expected.

2.2 Histatins are the Wound Closing Factors in Saliva

After EGF was ruled out as the main component for parotid induced woundclosure, RP-HPLC was used to separate parotid saliva and the activity ofthe pooled fractions was determined. First we found activity only in the2^(nd) fraction. In this fraction, the bulk of proteins present inparotid saliva, including amylase, is absent, which was confirmed by SDSpage gel electrophoresis. The 2^(nd) fraction could further be separatedinto two fractions, designated fraction 4 and 5. Fraction 5 was found tohave biological activity. Fraction 5 was further separated using a C18column, and consisted of 4 proteins, which were analyzed with ion trapmass spectrometry with a LCQ Deca XP (Thermo Finnigan). Biologicalactivity was solely found in designated peak 6. By ion trap mass MS itwas found that peak 6 contained a product of the Htn1 gene. Htn1 isresponsible for Histatins 1 and 2, and Htn2 for Histatins 3 and 5.

2.3 Potency of Histatin induced wound closure, stereo-specificity ofHistatin 2, candidacidal activities of the different histatins. Next thepotency of the different Histatins in both wound healing as theircandidacidal activities was studied. Histatins are known for theirantimicrobial activity. Histatin 5 generally has the lowest LC₅₀(concentration in which 50% of the micro-organisms are killed), and thekilling activity is reduced with increasing ionic strength.

We tested synthesized Histatins 1, 2, 3, 5 and the enantiomer ofHistatin 2 (designated D-Histatin 2) on wound closure and candidacidalactivities. In the test concentrations of 1, 5, 10, 30 and 50 μg/ml ofthe peptides were tested. Results shows that optimal concentration were,for Histatin 1, 10-30 μg/ml, for Histatin 2, 5-10 μg/ml, and forHistatin 3, 30-50 μg/ml. (Table 1). Histatins 1, 2 and 3 all inducewound closure, D-Histatin 2 and Histatin 5 do not.

Wound closure Candicidal activity rel. LC50 ± SD Peptide SequenceSEQ ID NO: clos. ± SD 1 mM PPB SFM Histatin 1DSHEKRHHGYRRKFHEKHHSHREFPFY SEQ ID NO: 4 1.22 ± 0.12*  6.0 ± 0.3 >100GDYGSNYLYDN Histatin 2 RKFHEKHHSHREFPFYGDYGSNYLYDN SEQ ID NO: 5 1.36 ±0.13* 13.8 ± 1.9 >100 D-Histatin 2 RKFHEKHHSHREFPFYGDYGSNYLYDNSEQ ID NO: 5 0.99 ± 0.14  10.7 ± 1.2 >100 Histatin 3DSHAKRHHGYKRKFHEKHHSHR..... SEQ ID NO: 6 1.21 ± 0.12*  1.1 ± 0.1 >100G.YRSNYLY DN Histatin 5 DSHAKRHHGYKRKFHEKHHSHR..... SEQ ID NO: 30 1.04 ±0.11   2.3 ± 0.1 >100 G.Y EGF 1.41 ± 0.12*

Histatin 2 has the most potent induction of wound closure, which doesnot significantly differ from the optimal EGF induced wound closure.Also, lowest concentration of Histatin 2 is needed to achieve thisclosure. Interestingly, D-Histatin 2 does not exhibit wound closureinduction, indicating at least partial stereo-specific activation ofprocesses inducing wound closure.

Also, Histatin 5 does not show induced wound closure, implying anecessity of parts of particular amino acids in the peptide.Contradictorily, the candidacidal activity in 1 mM PPB shows nostereo-specificity of Histatin 2 and point out Histatins 3 and 5 as themost potent, i.e. the lowest LC₅₀. Additionally, in SFM (150 mM) nocandidacidal activity whatsoever was could be demonstrated. Also, at anionic strength of 50 mM, which is in the physiological range of salivaalready proved enough to completely abolish the candidacidal effect ofhistatins (data not shown). Altogether the results of the experiments inTable 1 clearly show that the structural features involved in thecandidacidal of histatin 5 and wound healing properties of histatin 1,2and 3 are completely different.

2.4 Further Observations

In Candida Histatin 5 is internalized and associates with the energizedmitochondrion. Given the abovementioned discrepancy between C. albicansand epithelial cells we were very interested whether histatins becomeinternalized and where in the cells they accumulate. In co-accordancewith their wound healing properties Histatins 1, 2 and 3 areinternalized, and D-Histatin 2 and Histatin 5 are not (data not shown).NaN₃ inhibits the function of cytochrome c oxidase by bindingirreversibly to the heme cofactor. Thus, NaN₃ treatment lead us toconclude that the internalization energy dependent. Trypsin treatmentshows us that membrane proteins are essential for the internalization.Which, in addition to the stereo-specificity, is indicating a receptormediated process. We used mitochondria and acidic lysosome specificmarkers to elucidate histatin's fate once it is internalized

Erk1/2 Responsible for Histatin Induced Wound Closure

A mechanism has been proposed in which in vitro wound closure iscoordinated by two mitogen-activated protein kinase (MAPK) cascades. Thep38^(MAPK) cascade is responsible for migration and the extracellularsignal-regulated kinases (ERK1/2) cascade for proliferation.Furthermore, blockage of one activates the other, indicating cross-talk.Another paper, which affirmed this model, showed that epidermal growthfactor (EGF) stimulated migration required continuous presence of theligand. Of course it is very interesting to study which pathways areconducted when cells are stimulated with histatins. In FIG. 2 we affirmthe in FIG. 1 shown EGFR independent induced wound closure of saliva bydoing the same for Histatin 2. We also confirm the previously mentionedstudies, which show EGF induced wound closure is p38^(MAPK) dependent.Histatin 2 induced wound closure however, is not p38^(MAPK) but appearsERK1/2 dependent. These findings also suggest receptor mediatedactivation. Histatins are relatively cheap to manufacture, and thus canbe a target of becoming a clinical wound inducing substance.

Example 2 Testing of Various Peptides

Peptides were synthesized as described above and tested in the “woundclosure assay” (In vitro wound healing experiments) according to Example1 in a concentration of 10 μg/ml.

FIG. 3 shows both the peptides tested and the results obtained. Inaddition to the mentioned amino acid sequences, peptides were testedwherein the first four amino acids at the N-terminal were removes(RKFH). The removal thereof did not influence the activity in the assay.Results were compared to control. The results show that the amino acidsaccording to the invention show good activity in the assays.

Example 3 Cyclic Peptides

A cyclic peptide having the amino acid sequence according to SEQ.ID.NO.4, Histatin 1, was produced using a WANG LL resin available fromNovabiochem (EMD Biosciences), described in detail in Novabiochem Letter2/06 (EMD Biosciences Inc. P.O. Box 12087 La Jolla, Calif. 92039-2087).Cyclization of the linear peptide attached to the resin was according toGoncalves et. al. (Tetrahedron 61 (2005) 7789-7795). In short, aftercompletion of the peptide assembly, the resin was treated with 2%hydrazine/DMF at room temperature for 3 min. The treatment was repeatedtwo more times, and the partially protected resin was thoroughly washedwith DMF. For the intramolecular cyclization 0.3-3 M equiv of eachbenzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexa-fluorophosphate(PyBOP) and HOBt were used in the presence of 6 M equiv ofdiisopropylethylamine (DIEA) for 72 h. The obtained product wasfractionated by HPLC chromatography and the resulting fractions tested.

Next, the cyclic peptide was compared to the linear peptide in the invitro wound healing experiments as described under Example 1. Resultsare shown in FIG. 4.

FIG. 4 shows the relative wound closure activity of either the cyclicpeptide (C-Hst1) or the linear peptide (Hst1) at different concentrationof each of the peptides. Even at a concentration of 1*10−3 μM C-HST1,relative wound closure is still 1.5, whereas wound closure activity ofHST1 has dropped to just above 1 (Relative wound closure activity of 1is the wound closure activity when no peptide is added to the assaydescribed above). The results clearly show that cyclic peptidesaccording to the invention, having an amino acid sequence according tothe invention, show remarkably increased activity in comparison to thelinear peptides according to the invention, even when the concentrationis 5, 10 or even 100 times lower in comparison to the linear peptide.

1-35. (canceled)
 36. A composition comprising a peptide and at least onepharmaceutically acceptable excipient, wherein the peptide comprises atleast six contiguous residues of the amino acid sequence of SEQ ID NO: 5or a variant peptide thereof in which one amino acid residue issubstituted, deleted or inserted within said at least six contiguousresidues, wherein the peptide is a cyclic peptide.
 37. The compositionof claim 36, wherein the sequence of said at least six contiguousresidues is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2 and SEQ ID NO:
 3. 38. The composition of claim 36, wherein thesequence of said at least six contiguous residues comprises at least sixcontiguous residues of SEQ ID NO:
 2. 39. The composition of claim 36,wherein the cyclic peptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-29 or a variant thereof inwhich one amino acid residue in any of SEQ ID NOs: 4-29 is substituted,deleted or inserted and the one residue substitution, deletion orinsertion is not within a subsequence of SEQ ID NOs: 1-3.
 40. Thecomposition of claim 36, wherein the cyclic peptide has a relative woundclosure activity that is equal to the relative wound closure activity of1 μM concentration of the linear peptide of SEQ ID NO: 4 but at aconcentration of cyclic peptide that is at least 5 times lower.
 41. Thecomposition of claim 36, wherein the peptide has the sequence of SEQ IDNO:
 2. 42. The composition of claim 36, wherein the cyclic peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 4, 5, 7-20, 23-25, and 28-29 or a variant thereof inwhich one amino acid residue in any of SEQ ID NOs: 4, 5, 7-20, 23-25,and 28-29 is substituted, deleted or inserted and the one residuesubstitution, deletion or insertion is not within a subsequence of SEQID NO: 2.